The goal of this project is to elucidate structure-function relationships in macromolecular assemblies. During FY16, our studies focused on retinoschisin (RS1), a junctional protein in the human retina; and on encapsulin, a bacterial nanocompartment that sequesters iron. In prior work, we found that encapsulin forms icosahedral shells akin to viral capsids with their subunit assuming a fold previously encountered only in viral capsid proteins. 1) Retinoschisin protein (RS1) forms back-to-back octameric rings, suggesting a junctional model for cell-cell adhesion. RS1 is a protein required to maintain the structural and functional integrity of the retina. Mutations in RS1 lead to early vision impairment in young males, a condition called X-linked retinoschisis (XLRS), that is characterized by separation of inner retinal layers and disrupting synaptic signaling. From prior work in other laboratories, RS1 was thought to form an octamer, with each subunit comprising a discoidin (DS) domain and a small N-terminal (RS1) domain. We have used cryo-electron microscopy to determine the structure of RS1 at 0.4 nm resolution. In this way, we found that the complex consists, in fact, of two opposing octameric rings. In a ring, each subunit has the canonical discoidin fold. The RS1 domains occupy the centers of the rings, but are less clearly defined, suggesting mobility. We modeled the DS domains, consistent with intramolecular and intermolecular disulfides previously reported. The interfaces internal to and between rings feature residues implicated in XLRS, indicating the importance of correct assembly of the 16-meric complex to obtain a correctly constituted junction. Loops at the periphery of the rings may bind sugars and lipids on the membrane surface. As RS1 is entirely extracellular and without membrane-embedded loops, it apparently couples neighboring retinal membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components. The paper reporting these observations was recently published (Tolun et al, 2016). 2) A virus capsid-like nanocompartment that stores iron and protects bacteria from oxidative stress. Living cells compartmentalize materials and enzymatic reactions to increase their metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely to a large extent on protein-bound nanocompartments. Encapsulins are a recently discovered class of nanocompartments. Hitherto, their functions have been unclear. We have characterized the structure of encapsulins isolated from the anaerobe from Myxococcus xanthus and shown that its role is to sequester cytosolic iron, thereby to protect the cells from oxidative stress. This nanocompartment consists of a protein shell with internal contents. It has a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-EM, we determined that EncA expressed in E. coli self-assembles into an icosahedral shell 32 nm in outer diameter built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (30,000 Fe atoms vs. 3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase five-fold upon starvation, protecting cells from oxidative stress through iron sequestration. These results were published in FY14 (McHugh et al., 2014). Since then, we have been investigating this system further, in several directions: determining the crystal lattice spacings in the iron-phosphate granules by electron diffraction; identifying the crystal form of mineralization; expressing the internal proteins with an aim to crystallographic studies; and extending the resolution of cryo-EM analyses. We plan to express, purify and crystallize the three cargo proteins, starting with EncB, Preliminary cryo-electron microscopy data have been collected for the purified EncB protein, and crystallization trials are underway, with findings that suggest an incomplete ferritin-like fold according to bioinformatic analysis. To probe the structure of the mineralized iron we have carried out preliminary electron diffraction experiments on iron-containing encapsulins and observed discrete reflections. We are also testing if iron can be released from encapsulin particles in vitro, considering that the bacterium might have a need for cytosolic iron under certain environmental conditions. 3) Further development of image processing software for three-dimensional electron microscopy has continued. An ongoing initiative in the EM community is the development of guidelines and standards to ensure ease-of-use of software and to promote confidence in the quality of the results. Conventions for the interchange of information for single particle analysis (SPA) to mitigate the complications that arise from using multiple software packages (Marabini et al, 2016). The intent is to extend these conventions to cover more situations and processing techniques, including tomography. Another part of this effort is to make sure that common parameters have unambiguous meanings. The Contrast Transfer Function (CTF) challenge was an effort to compare CTF parameters as determined from different micrographs and between different software packages. This has led to a better understanding of the difficulties in estimating CTF parameters and how the software packages deal with them. Moreover, the outcome of a SPA analysis can be influenced by the investigator if care is not taken to avoid bias. To counter this, a validation principle was proposed, based on the idea that a valid reconstruction would require fewer particle images compared to a reconstruction from aligned noise images (Heymann, 2015).